中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (25): 3987-3992.doi: 10.3969/j.issn.2095-4344.1778

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

人脐血间充质干细胞对小鼠骨髓巨噬细胞M2亚型的转化作用

陈柄全，彭  漪，肖  轶，彭智勇，赵吉玲，余国龙
  

  1. 中南大学湘雅医院心内科，湖南省长沙市  410008
  • 修回日期:2019-03-10 出版日期:2019-09-08 发布日期:2019-09-08
  • 通讯作者: 余国龙，博士，教授，中南大学湘雅医院心内科，湖南省长沙市 410008
  • 作者简介:陈柄全，男，1991年生，湖南省张家界市人，土家族，2018年中南大学湘雅医学院毕业，硕士，医师，主要从事干细胞治疗心肌梗死的基础医学研究。
  • 基金资助:

    国家自然科学基金面上项目(81570266)，项目负责人：余国龙；湖南省自然科学基金重点项目(2015SK2025)，项目负责人：余国龙

Effect of human umbilical cord blood mesenchymal stem cells on the transformation of M2 macrophages from mouse bone marrow

Chen Bingquan, Peng Yi, Xiao Yi, Peng Zhiyong, Zhao Jiling, Yu Guolong
  

  1. Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • Revised:2019-03-10 Online:2019-09-08 Published:2019-09-08
  • Contact: Yu Guolong, MD, Professor, Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • About author:Chen Bingquan, Master, Physician, Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • Supported by:

    the National Natural Science Foundation of China (General Program), No. 81570266 (to YGL); the Natural Science Foundation of Hunan Province, No. 2015SK2025 (to YGL)

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摘要：

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文题释义：
人脐血间充质干细胞具有更多的优势：①脐血获取方法较骨髓更容易，无任何痛苦和不良作用，更易让人们接受；②与骨髓来源间充质干细胞没有明显的形态学和免疫表型的差异；③相较于骨髓来源，具有更强的繁殖能力；④收集的脐血更容易保存，可以放在液氮中长期保存，复苏后可以维持90%的细胞活性；⑤使用人脐血在伦理学上更容易被人们所接受；⑥现在各国已经建立起脐血库，为干细胞治疗奠定坚实的基础。
M1/M2型巨噬细胞：巨噬细胞是非特异性免疫的重要组成部分，在炎症和宿主防御中发挥重要作用。在组织中，巨噬细胞可以因为环境信号(例如微生物产物、受损细胞、活化的淋巴细胞)表达出不同的免疫表型。两种功能及表型相反的巨噬细胞被命名为经典活化的巨噬细胞(M1型)和交替活化型巨噬细胞(M2型)。M1型巨噬细胞生物特性是杀菌活性加强，并产生多种促炎的递质；而M2型巨噬细胞主要生物特性是促进炎症吸收与消退，产生多种抑炎的递质，促进炎性组织修复。

 

摘要
背景：间充质干细胞已经被用于多种疾病的治疗，且研究表明间充质干细胞可能是通过促进巨噬细胞向M2亚型转化来产生生物效应，但这种效应是否呈量效、时效关系及其转化效应是否需要细胞间直接接触尚不明确。
目的：验证人脐血间充质干细胞对巨噬细胞M2亚型转化效应，并探讨人脐血间充质干细胞对巨噬细胞M2亚型转化效应的剂量与时间相关性。
方法：从小鼠骨髓中提取单核细胞，采用巨噬细胞集落刺激因子诱导产生M0型巨噬细胞(F4/80+，CD11B+)。将M0型巨噬细胞以1×106/孔密度植入Transwell板或6孔培养板中，分别以不同密度(0，2×105，4×105)人脐血间充质干细胞在含有脂多糖(100 μg/L)/干扰素γ(20 μg/L)完全培养基中直接或间接共培养24，48 h。显微镜下观察巨噬细胞形态学改变，流式细胞仪检测M2型巨噬细胞(CD206+，CD11C-)占整体比例，ELISA检测细胞培养上清液白细胞介素10、白细胞介素6和白细胞介素1β水平。
结果与结论：①与人脐血间充质干细胞共培养后，巨噬细胞触角伸长，呈长索状M2型细胞形态特征；②与人脐血间充质干细胞直接共培养、间接共培养24，48 h，M2型巨噬细胞百分比显著性增加(P < 0.05)；③人脐血间充质干细胞低浓度与高浓度组间比较，M2型巨噬细胞百分比差异无显著性意义；④直接或间接培养48 h的M2型巨噬细胞百分比均显著高于24 h，差异有显著性意义(P < 0.05)。⑤人脐血间充质干细胞直接共培养、间接共培养上清中促炎性细胞因子白细胞介素6、白细胞介素1β水平显著减少，而抑炎性细胞因子白细胞介素10水平显著增加(P < 0.05)；⑥直接或间接共培养48 h抑炎性细胞因子白细胞介素10水平均较24 h显著上升(P < 0.05)；⑦结果表明，人脐血间充质干细胞与巨噬细胞直接与间接接触均促进巨噬细胞M2亚型转化，其转化效应有时效依赖性，而与细胞浓度无相关性。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-4147-1461(陈炳全)

关键词: 人脐血间充质干细胞, 巨噬细胞, 巨噬细胞M1/M2亚型, M2亚型转化效应, 炎症反应, 直接共培养, 间接共培养, 国家自然科学基金

Abstract:

BACKGROUND: Mesenchymal stem cells have been used in the treatment of a variety of diseases. They may produce biological effects by promoting the conversion of macrophages to M2 subtypes, but it is unclear whether these effects are dose- and time-sensitive, as well as whether intercellular direct contact is involved in the cell transformation.
OBJECTIVE: To verify the effect of umbilical cord blood mesenchymal stem cells (hUC-MSCs) on the transformation of M2 subtypes of macrophages, and to investigate the dose- and time-dependent effects of hUC-MSCs on the transformation of M2 subtypes of macrophages. 
METHODS: Monocytes were extracted from mouse bone marrow and induced by macrophage colony-stimulating factor to produce M0 macrophages (F4/80+, CD11B+). M0 macrophages were implanted into Transwen plates or 6-well culture plates at a density of 1×106/well, and co-cultured directly or indirectly with hUC-MSCs at different concentrations (0, 2×105, 4×105) in complete medium containing lipopolysaccharide (100 μg/L) and interferon-γ (20 μg/L) for 24 and 48 hours. The morphological changes of macrophages were observed under direct microscope. The number of M2 macrophages (CD206+, CD11C-) was detected by flow cytometry. The levels of interleukin-10, interleukin-6 and interleukin-1β in the cell culture supernatant were measured by ELISA. 
RESULTS AND CONCLUSION: (1) After co-cultured with hUC-MSCs, the macrophages were elongated in cell antennae and the funicular cells, like M2 macrophages, appeared. (2) After 24 and 48 hours of direct or indirect co-culture with hUC-MSCs, the percentage of M2 macrophages was significantly increased (P < 0.05). (3) The percentage of M2 macrophages insignificantly differed after direct or indirect co-culture with low- and high-concentration hUC-MSCs. (4) The percentage of M2 macrophages was significantly higher after 48 hours co-culture than after 24 hours co-culture (P < 0.05). (5) After direct or indirect co-culture with hUC-MSCs, the levels of interleukin 6 and interleukin-1β in the cell supernatant were decreased significantly, while the interleukin-10 level increased significantly (P < 0.05). (6) The interleukin-10 level was significantly increased after 48 hours co-culture relative to that after 24 hours co-culture. In conclusion, hUC-MSCs can promote the transformation of M2 macrophages in a time-dependent but not concentration-dependent manner.

Key words: human umbilical cord blood mesenchymal stem cells, macrophages, macrophage M1/M2 subtype, M2 subtype transformation, inflammatory response, direct co-culture, indirect co-culture, National Natural Science Foundation of China

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